THE ROLE OF THE PATHOLOGIST IN
DRUG DISCOVERY AND DEVELOPMENT
- TARGET VALIDATION
Dr Dinah Parums, Principal Pathologist
...
IMMUNOHISTOCHEMISTRY and
IMMUNOFLUORESCENCE
 Tissue sections from
normal and diseased
specimens on glass
slides as whole
...
WHAT CAN Immunohistochemistry (IHC)
SHOW ? ?



The detection of
target antigens
(usually proteins)
within tissues and
ce...
WHAT CAN IHC (and
IMMUNOFLUORESCENCE) SHOW ?
Double confocal
immunofluorescence

McAb CD31
localizing
to endothelial cells...
CONSIDERATIONS FOR ANTIBODY USE
 ‘Clean’ monoclonal and polyclonal antibodies
should be used (confirmed by western blot o...
6 KEY STEPS FOR IHC
IN TUMOUR BIOMARKER DEVELOPMENT

1 NEED FOR SPECIFIC ANTIBODY
TO IDENTIFY PROTEIN MARKER.

Custom made...
NON ISOTOPIC IN SITU HYBRIDIZATION
(NISH) STUDIES











ISH assays allow for the detection of nucleic
acid ...
NON ISOTOPIC IN SITU HYBRIDIZATION
(NISH)
Like antibodies, each probe must be individually
optimized for reactivity in tis...
BENEFITS OF IHC AND NISH ASSAYS
 Specific, high
resolution
detection of
targets in human
tissue
 Maintenance of
tissue m...
BENEFITS OF IHC AND NISH ASSAYS

Breast cancer peri-tumour angiogenesis.
NISH using a digoxygenin labelled TGFbeta
ribopro...
BENEFITS OF IHC AND NISH ASSAYS
 Human tissue is
more likely to
reflect ‘real life’
expression of a
target than cell
line...
Laser Capture Microdissection for Molecular Analysis

Pixcell II system
Expert pathologists
Transcription biologists
Befor...
Expression Profiles in Clinical Series: Tissue Microarrays

0.3 mm tissue
cores

Immunohistochemistry
TMA construction
eg....
Applications of Tissue Microarrays
(TMAs)









Characterisation of new
antibodies for IHC
Gene expression
profil...
The Future of Histopathology The Concept of ‘Pathology IT’ and
Individualised Diseased ‘Tissue Profiling’









...
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Dr Dinah Parums. The Role of The Pathologist in Target Identification and Validation in Targeted Therapy and Personalized Medicine.

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Dr Dinah Parums. The Role of The Pathologist in Target Identification and Validation in Targeted Therapy and Personalized Medicine.

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Dr Dinah Parums. The Role of The Pathologist in Target Identification and Validation in Targeted Therapy and Personalized Medicine.

  1. 1. THE ROLE OF THE PATHOLOGIST IN DRUG DISCOVERY AND DEVELOPMENT - TARGET VALIDATION Dr Dinah Parums, Principal Pathologist  Biotechnology and pharmaceutical companies are challenged to validate the pool of potential drug targets and determine those most appropriate to enter a drug development programme.  A valuable method of target validation is their localisation to specific cells and tissues using immunohistochemistry (IHC) and non isotopic in situ hybridisation (NISH) techniques pinpointing the expression of protein and nucleic acids respectively.
  2. 2. IMMUNOHISTOCHEMISTRY and IMMUNOFLUORESCENCE  Tissue sections from normal and diseased specimens on glass slides as whole sections, multiblocks or TMAs  Tissues are frozen or formalin fixed and embedded in paraffin wax  Formalin fixed tissues offer better morphology and are more readily available but fixation must be standardised
  3. 3. WHAT CAN Immunohistochemistry (IHC) SHOW ? ?  The detection of target antigens (usually proteins) within tissues and cells  Relative level of target expression  Subcellular localisation of the target (nuclear, cytoplasmic, cell membrane) McAb ASMA in myofibroblasts in healing skin McAb ASMA in myofibroblasts in healing skin Confocal immunofluorescence
  4. 4. WHAT CAN IHC (and IMMUNOFLUORESCENCE) SHOW ? Double confocal immunofluorescence McAb CD31 localizing to endothelial cells (Red) McAb ASMA localizing to smooth muscle cells (Green)
  5. 5. CONSIDERATIONS FOR ANTIBODY USE  ‘Clean’ monoclonal and polyclonal antibodies should be used (confirmed by western blot or immunoprecipitation)  Polyclonal antibodies should be affinity purified  Antibodies generated from peptides or complete proteins can be used  Binding of an antibody to a target in tissues is empirical thus each antibody should be tested separately for reactivity in tissues Polyclonal antibody to TGF beta in infiltrating lobular carcinoma of the breast localises to stromal spindle cells and collagen. Immunoperoxidase with DAB. Is this specific or not ?
  6. 6. 6 KEY STEPS FOR IHC IN TUMOUR BIOMARKER DEVELOPMENT 1 NEED FOR SPECIFIC ANTIBODY TO IDENTIFY PROTEIN MARKER. Custom made Commercially available Academic institution 2 TEST FOR SPECIFICITY IN HUMAN/ANIMAL TISSUE. 3 TEST FOR EFFECT OF CANDIDATE DRUG (CD) ON PROTEIN EXPRESSION IN TUMOUR XENOGRAFT IN ANIMAL MODEL. 4 FEASIBILITY AND VARIABILITY ASSESSMENT IN NORMAL AND HUMAN TUMOUR TISSUE (fresh, frozen or formalinfixed). 5 EFFECT OF CD ON NORMAL AND TUMOUR TISSUE (fresh, frozen or formalin-fixed). PROOF OF MECHANISM/PROOF OF PRINCIPLE. 6 EFFECT OF CD ON NORMAL AND TUMOUR TISSUE (fresh frozen or formalin fixed) AND LINKED TO DISEASE OUTCOME. PROOF OF CONCEPT.
  7. 7. NON ISOTOPIC IN SITU HYBRIDIZATION (NISH) STUDIES           ISH assays allow for the detection of nucleic acid sequences in cells and tissues. The target molecules are specific mRNA sequences RNA and DNA probes can be used For riboprobes it is common to make both sense and antisense probes The antisense probe is a complementary strand to the target mRNA and should bind with it Generally, riboprobes are chosen rather than oligonucleotide or DNA probes The ideal probe length is debatable but 200 to 500 bases work well. Longer probes may be more sensitive but can be difficult to hybridize to their targets Radioisotopically labelled probes are very sensitive but yield lower resolution with results being more difficult to assess Biotinylated probes may be problematic due to endogenous biotin and biotin-binding proteins
  8. 8. NON ISOTOPIC IN SITU HYBRIDIZATION (NISH) Like antibodies, each probe must be individually optimized for reactivity in tissues, with the variables to consider including; Breast cancer peri-tumour angiogenesis. NISH using a digoxygenin-labelled VEGF riboprobe  Probe length  Probe labelling  Probe concentration  Protease concentration  Hybridization conditions  Stringency washes  Detection methodology
  9. 9. BENEFITS OF IHC AND NISH ASSAYS  Specific, high resolution detection of targets in human tissue  Maintenance of tissue morphology  Histopathological identification  Identification of cell types  Comparison of Breast cancer peri-tumour angiogenesis. NISH using a digoxygenin-labelled TGFbeta riboprobe normal and diseased tissue localises to lymphocytes (Blue). IHC using a APAAP and Fast Red and CD31 localises to endothelial cells (Red).
  10. 10. BENEFITS OF IHC AND NISH ASSAYS Breast cancer peri-tumour angiogenesis. NISH using a digoxygenin labelled TGFbeta riboprobe and APAAP/Fast Red localises to lymphocytes (Red). IHC using immunoperoxidase and DAB CD31 localises to endothelial cells (Brown).  In cases where a celltype may not be recognised by morphology alone, multiple labelling studies can be used  The use of quality controlled reagents and techniques yields consistent and reproducible results  Paraffin-embedded specimens allow for retrospective studies
  11. 11. BENEFITS OF IHC AND NISH ASSAYS  Human tissue is more likely to reflect ‘real life’ expression of a target than cell lines  In ‘grind and find’ assays (such as western and northern blots) tissue architecture is lost and so is Breast cancer peri-tumour angiogenesis. cell-specific NISH using a digoxygenin labelled TGFbeta riboprobe resolution and APAAP/Fast Red localises to lymphocytes (Red).  A target expressed Immunofluorescence with McAb CD31 localises to at high levels by endothelial cells (Green). Endothelial cells expressing cells which are TGFbeta = Yellow. present in small
  12. 12. Laser Capture Microdissection for Molecular Analysis Pixcell II system Expert pathologists Transcription biologists Before DNA Mutation analysis DNA fingerprinting Capture After RNA cDNA microarrays Protein Proteomics 15 1 4 23 11 16 7 9 12 8 22 10 13 5 6 23 24 14 17 18 19 20 21
  13. 13. Expression Profiles in Clinical Series: Tissue Microarrays 0.3 mm tissue cores Immunohistochemistry TMA construction eg. Breast cancer Pathology input In Situ Hybridisation
  14. 14. Applications of Tissue Microarrays (TMAs)       Characterisation of new antibodies for IHC Gene expression profiling for differential diagnosis Gene expression profiling for carcinoma of unknow n primary site Gene expression profiling for molecular subclassification of tumours Array based comparative genomic hybridisation (ACGH) for differential diagnosis Gene expression profiling and/or ACGH for identification of molecular therapeutic targets with the goal of achieving individualised GENE ARRAYS TISSUE ARRAYS • • one sample many markers many samples one marker • Gene expression Antibodies • Gene Amplification/d eletion In situ hybridisation
  15. 15. The Future of Histopathology The Concept of ‘Pathology IT’ and Individualised Diseased ‘Tissue Profiling’        Automated Histopathology, IHC, NISH and Image Analysis Multiple IHC markers on one slide Combined IHC and in-situ RNA profiling In situ detection of multiple RNA transcription sites (using NISH or FISH) Multivariate analysis of imaging and protein and mRNA expression Disease/tumour profiling for the individual patient with predictive and prognostic implications, predictive information regarding drug responses Implications for future clinical trials work

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